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<div class='MSDStitle'>Materials</div>
		<table width='100%'>
          <tr>
		    <td width='35px' valign='top'></td>
			<td valign='top'>
			  <ul>
				<li><span style='font-weight:bold;'>2x PCR Taq Mastermix</span> (25ul per reaction)</li>
				<li><span style='font-weight:bold;'>Mycoplasma PCR Primer Mix</span> (1ul per reaction)</li>
				<li><span style='font-weight:bold;'>Mycoplasma Positive Control</span> (1ul per control reaction)</li>
                <li><span style='font-weight:bold;'>Cell Culture Supernatant</span> (1ul per test reaction)</li>
                <li><span style='font-weight:bold;'>ddH<sub>2</sub>O</span> (23-24ul per reaction)</li>
			  </ul>
			</td>
		  </tr>
		</table>
	  <div class='MSDStitle'>Protocol</div>
		<table width='100%'>
		  <tr>
		    <td width='35px' valign='top'>&nbsp;</td>
		    <td valign='top'>
			  <ol>
				<li>The prospective contaminated cells should be in culture for at least 24 hours prior to mycoplasma detection.</li>
				<li>Aliquot 0.5ml of prospective contaminated cell culture medium and centrifuge the sample for 5 minutes at 2000x<em>g</em> to precipitate cells and debris. Use the cell culture supernatant as the test sample.</li>
                <li>Prepare the reactions according to the following table:</li>
                	<table id='myco' width='93%'>
                    	<tr>
                        	<th>&nbsp;</th>
                            <th>Test Sample</th>
                            <th>Positive Control</th>
                            <th>NTC Control</th>
                        </tr>
                        <tr>
                        	<th>2x PCR Taq Mastermix</th>
                            <td>25ul</td>
                            <td>25ul</td>
                            <td>25ul</td>
                        </tr>
                        <tr>
                        	<th>Mycoplasma PCR Primer Mix</th>
                            <td>1ul</td>
                            <td>1ul</td>
                            <td>1ul</td>
                        </tr>
                        <tr>
                        	<th>Cell Culture Supernatant</th>
                            <td>1ul</td>
                            <td>-</td>
                            <td>-</td>
                        </tr>
                        <tr>
                        	<th>Mycoplasma Positive Control</th>
                            <td>-</td>
                            <td>1ul</td>
                            <td>-</td>
                        </tr>
                        <tr>
                        	<th>ddH<sub>2</sub>O</th>
                            <td>23ul</td>
                            <td>23ul</td>
                            <td>24ul</td>
                        </tr>
                        <tr>
                        	<th>Total Volume</th>
                            <td>50ul</td>
                            <td>50ul</td>
                            <td>50ul</td>
                        </tr>
                    </table> 
                  <li>Perform 30-40 cycles of PCR amplification as follows:</li>
                    <table id='myco' width='93%'>
                    	<tr>
                        	<th>&nbsp;</th>
                            <th>Temperature</th>
                            <th>Duration</th>
                            <th>Cycles</th>
                        </tr>
                        <tr>
                        	<th>Enzyme Activation</th>
                            <td>95°C</td>
                            <td>5 minutes</td>
                            <td>Hold</td>
                        </tr>
                        <tr>
                        	<th>Denature</th>
                            <td>95°C</td>
                            <td>30 seconds</td>
                            <td rowspan="3">30-40</td>
                        </tr>
                        <tr>
                        	<th>Anneal</th>
                            <td>55°C</td>
                            <td>30 seconds</td>
                        </tr>
                        <tr>
                        	<th>Extend</th>
                            <td>72°C</td>
                            <td>1 minute</td>
                        </tr>
                        <tr>
                        	<th>Final Extension</th>
                            <td>72°C</td>
                            <td>10 minutes</td>
                            <td>1</td>
                        </tr>
                        <tr>
                        	<th>Holding</th>
                            <td>4°C</td>
                            <td>-</td>
                            <td>Hold</td>
                        </tr>
                    </table> 
                  <li>Analyze the ampliciation products by agarose gel electrophoresis and visualize by ethidium bromide or SafeView (Cat. No. G108) staining. Since the PCR Taq Mastermix already contains a loading dye, further addition of loading dye is not required. For DNA fragment length confirmation, the 100bp plus OptiDNA Marker (Cat. No. G193) is recommended.</li>
                  <li>The presence of PCR products approximately 500bp in length indicates the presence of mycoplasma contamination in the original cell culture. Depending on the different mycoplasma type, the length of the PCR product will vary between 450bp to 550bp.    
              </ol>
			</td>
		  </tr>
		</table>